Project Summary/Abstract Glycoproteomics is the large-scale study of carbohydrate modifications on proteins, including site-specific glycan heterogeneity. The most commonly used platform for glycoproteomics is liquid chromatography, electrospray-ionization tandem mass spectrometry (LC-ESI-MS/MS), the same platform used for proteomics, but glycoproteomics is inherently more difficult than proteomics for a variety of reasons. Glycopeptide signals are split over many glycoforms, so they tend to be less intense than ordinary peptide signals. Glycopeptides ionize less easily and produce lower-quality tandem mass spectra, because it is difficult to simultaneously fragment both the peptide and glycan parts. Finally, glycopeptide data analysis is much more difficult than ordinary peptide data analysis, due to the explosion of possibilities, for example, 10s to 100s of glycan compositions per N-linked glycosylation site, and fewer compositions but much more closely spaced sites in the case of O-linked glycosylation. Despite these challenges, glycoproteomics is becoming increasingly feasible with improvements in experimental methods (for example, zinc-finger knockouts), data acquisition modes (such as electron-transfer dissociation), and data analysis software (Byonic search engine). With these improvements and others, glycoproteomics has the potential to leverage the vast worldwide investment in proteomics to revolutionize glycobiology. In this project, we propose to accelerate development of Byonic, the only general-purpose proteomics search engine with special support for glycopeptide analysis. Specifically, we propose integrated analysis of complementary fragmentation modes, integrated analysis of MS1 and MS2 (that is, intact and fragmented peptides), and finally topology (?cartoon?) assignment for glycopeptide spectra. Glycosylation plays many key biological roles, including cancer markers, immune system recognition, and sperm/egg binding, so worldwide improvement in glycoproteomics could have great impact for human health.